The main objective is the application of the RNA/DNA oligonucleotide technology to a gene therapy for rheumatoid arthritis by inactivating cytokines such as IL-alpha/beta and TNFalpha at their genetic level, either by incorporating a stop codon or causing a trans- dominant mutation in tissue culture cells. In contrast to the current gene therapy utilizing viral vectors, this proposal investigates an alternative approach, an oligonucleotide-based gene therapy by homologous recombination and DNA repair, potentially capable of reducing IL-1alpha/beta and TNFalpha directly in the synovium. The current grant proposal is focused on "fine-tuning" of the design and delivery system of oligonucleotides and on testing the feasibility of inactivation of IL-1alpha/beta and TNFalpha in human tissue culture cells, first in the established monocyte/macrophage cell lines, second in cells isolated from the synovium, third in synovium explants from RA patients. Gene therapy capable of introducing a gene or genes that code for proteins with therapeutic properties offers an attractive alternative to the traditional rheumatoid arthritis therapy. Since therapeutic agents are synthesized directly at the site of disease, delivery is no longer an issue. In addition, the gene transfer approach addresses the chronic nature of the diseases. Several gene therapies have been developed to antagonize IL -1- induced events utilizing a retroviral or adenoviral vector driven expression of the IL- 1 receptor antagonist. It has been difficult to sustain a high level of expression of the IL-1 receptor antagonist which is required for successful therapy. Moreover, there is a safety concern in using viral vectors. Thus, a nucleic-acid-based gene therapy which directly reduces IL -1 in the synovium may have some advantage over the current gene therapy strategies that antagonize the IL-1 effect indirectly. A novel experimental strategy has been developed which centers around site-specific correction of mutations using unique chimeric ODNs containing both RNA and DNA. A chimeric ODN consists of complementary sequences that are folded into a duplex with double-hairpin caps. One strand contains two RNA stretches interrupted with the DNA residues which are designed to make the mismatch to the targeted DNA. The RNA stretches of the chimeric ODN facilitates the homologous recombination, and the mismatch created between the targeted strand and chimeric oligonucleotide is repaired by the cellular DNA repair system. It has been demonstrated that chimeric ODNs enabled a correction of a mutant base in an extrachromosomal and chromosomal target in a sequence- specific and inheritable manner. A high frequency of gene correction, approaching 30% was observed at a low concentration of the chimeric oligonucleotide. The high efficacy and specificity of the chimeric oligonucleotide makes it applicable toward a gene therapy for correction of mutations found in human genetic diseases as well as toward a gene therapy for inactivation of harmful cellular proteins in a localized area, proposed in this grant application.